In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

Summary

Selectively labeled fluorescent GnRH-I, -II and -III derivatives are reliable tools for tracking and quantifying their cellular uptake. This manuscript introduces experiments to visualize, quantify and compare the uptake efficiency of these GnRH conjugates in various cell lines.

Abstract

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH receptors. However, the quantitative analysis of GnRH analogues' cellular uptake and the investigated cell types in GnRH-based drug delivery systems are currently limited. Previously introduced, selectively labeled fluorescent GnRH I, -II and -III derivatives provide great detectability, and they have suitable chemical properties for reproducible and robust experiments. We also found that the appropriate up-to-date methods with these labeled GnRH analogues could offer novel information about the GnRH-based drug delivery systems. This manuscript introduces some simple and fast experiments regarding the cellular uptake of [D-Lys⁶(FITC)]-GnRH-I, [D-Lys⁶(FITC)]-GnRH-II and [Lys⁸(FITC)]-GnRH-III on the EBC-1 (lung), the BxPC-3 (pancreas) and on the Detroit-562- (pharynx) malignant tumor cells. In parallel with these GnRH-FITC conjugates, the cell surface level of GnRH-I receptors was also examined on these cell lines before and after the GnRH treatment by confocal laser scanning microscopy. The cellular uptake of GnRH-FITC conjugates was quantified by fluorescence-activated cell sorting. In these experiments minor differences among GnRH analogues and major differences among cell types was observed. The significant differences among cell lines are correlated with their distinct level of cell surface GnRH-I receptors. The introduced experiments contain practical methods to visualize, quantify and compare the uptake efficiency of GnRH-FITC conjugates in a time- and concentration-dependent manner on various adherent cell cultures. These results could predict the drug targeting efficiency of GnRH conjugates on the given cell culture, and offer a good basis for further experiments in the examination of GnRH-based drug delivery systems.

Introduction

Peptide based targeted drug delivery systems have become a fast developing and promising area in cancer therapy over the past few years^1,2. Human gonadotropin releasing hormone receptor type I (GnRH-I-R) is primarily located in the pituitary gland but is also present in several other tissues which are responsible for self-reproduction³. GnRH-I-R is also expressed in a number of cancer tissues, related or unrelated to the reproductive system^4,5. The high expression of GnRH-I-R on several malignant tumor cells compared with healthy tissues provides an opportunity for targeted therapy^5,6.

Many gonadotropin-releasing hormone (GnRH) analogues have been developed in the last few decades, which could be used as targeting moieties^7,8,9. These peptides are able to deliver anticancer agents with high selectivity into malignant tumor cells which over-express GnRH-I-R⁶. Several GnRH conjugated anti-tumor drugs with higher selectivity and better efficiency than the corresponding unconjugated free drug have been reported^7,8,9.

Previous publications about GnRH peptides and their receptors reported that the GnRH-I-R can assume various conformations which have different selectivity for GnRH analogues¹⁰. The highly variable GnRH-I-R has complex and various signaling pathways are endowed with different activity against their natural and artificial ligands¹¹. These facts make investigation of GnRH-based systems challenging. On the other hand, they possess promising therapeutic potential. Several experiments with radiolabeled GnRH peptides were previously reported^12,13,14,15, but experiments in which fluorescently labeled GnRH analogues were used are still limited. While radioactive labeling offers high sensitivity, fluorescent labeling has several other advantages, for example the easier handling, and the ability to counterstain with different fluorophores. Three common GnRH analogues which have successfully been used for drug delivery are the [D-Lys⁶]-GnRH-I, [D-Lys⁶]-GnRH-II and GnRH-III, but the effectiveness of these peptides as targeting moieties is rarely compared^16,17. On the other hand, results from separate experiments in which different cancer cells and GnRH analogues were used is diverse.

Based on these considerations, we focused on the tumor targeting and drug delivery potential of these GnRH peptides, and thereby synthesized and characterized the [D-Lys⁶(FITC)]-GnRH-I, [D-Lys⁶(FITC)]-GnRH-II and [Lys⁸(FITC)]-GnRH-III peptide conjugates¹⁸. These analogues are selectively labeled with FITC on the side chain of their Lys or D-Lys (peptide-FITC ratio 1:1 at each conjugate). The idea was that the selective fluorescent labeling can offer novel information about these peptides, and allows their good tracking and reliable quantification. These conjugates have safe handling and reliable detectability, which make it easier to compare their tumor targeting efficiency, and the screening of numerous types of malignant tumor cells. We hope that up-to date experiments with these peptide conjugates could contribute to the development of novel cancer targeting GnRH-drug conjugates, and help to identify new therapeutic targets as well.

The present manuscript demonstrates some well reproducible and fast experiments with GnRH-FITC conjugates. The cell surface expression of GnRH-R is a determinative condition regarding GnRH uptake, therefore we simultaneously investigated the cell surface level of GnRH-I-R on the tested cell lines. We visualized the GnRH-I-R and GnRH-FITC conjugates by confocal laser scanning microscopy (CLSM) and quantified the cellular uptake of GnRH-FITC conjugates using fluorescence-activated cell sorting (FACS).

Protocol

1. Preparation of Cell Cultures and Reagents

1. Maintain the cell cultures in the manufacturer's recommended medium, supplemented with 10% (v/v) fetal bovine serum and antibiotics (called complete medium). Keep the cell culturing flask in a humidified, 5% CO₂ atmosphere incubator at 37 °C. Follow the proliferation and confluency of cells by inverted microscope (using 10X phase contrast objective).
2. When cells reach adequate confluency, remove the medium, and wash the culture with 2-3 mL, sterile phosphate-buffered saline (PBS). Remove the PBS and add 0.5 mL, sterile 0.25% trypsin-EDTA solution to the cell culture and incubate at 37 °C until cells detach (approximately 10 min).
3. Suspend the cells in 3-4 mL sterile complete medium to stop trypsin and transfer them into a sterile centrifuge tube. Centrifuge the cells at 150 x g for 4 min at room temperature (RT). Discard the supernatant carefully and suspend the pellet in 2-3 mL sterile complete medium.
4. Take out 100 µL of the cell suspension and mix with 100 µL 0.4% (m/V) trypan blue solution, to stain the dead cells. Load 10 µL of this mixture into a hemocytometer and determine the number of viable cells by inverted microscope.
5. Dilute the required amount of cell suspension prepared in step 1.3 to 10 mL with sterile complete medium, containing 4 x 10⁴ cell/mL.
6. Add 250 µL of cell suspension (prepared in step 1.5) per well (10⁴ cell/well) into seven wells of the first glass bottom 8-well microscopic slide. Use this slide in the GnRH uptake method.
7. Add 250 µL of cell suspension (prepared in step 1.5) per well (10⁴ cell/well) into five wells of the second 8-well microscopic slide. This slide will be used in the GnRH-I-R expression method.
8. Add 1 mL of cell suspension (prepared in step 1.5) per well (4 x 10⁴ cell/well) into seven wells of a 12-well plate. This plate will be used in the GnRH quantification method.
9. Let the cells adhere on the two microscopic slides and the plate. Incubate them in a humidified, 5% CO₂ atmosphere incubator at 37 °C for 48 h.
10. After the incubation check the cells by inverted microscope (using 10X phase contrast objective). If the cells are healthy and attached, continue with the following steps.
11. Prepare 10 mM GnRH-FITC stock solution in dimethyl sulfoxide (DMSO) from each of the three conjugates¹⁸. (Use these dilution concentrations: 16.43 µg/µL [D-Lys⁶(FITC)]-GnRH-I, 16.97 µg/µL [D-Lys⁶(FITC)]-GnRH-II and 16.47 µg/µL [Lys⁸(FITC)]-GnRH-III) Keep these stock solutions in a dark place at RT and use them up within a few weeks.
12. Dilute 1.7 µL each of the three 10 mM GnRH-FITC stock solutions in 1.7 mL complete medium. Shake these solutions. (These are the 10 µM GnRH-FITC treating media.) Dilute 150 µL each of the three 10 µM GnRH-FITC treating medium in 1.35 mL complete medium. Shake these solutions as well (these are the 1 µM GnRH-FITC treating medium).
    NOTE: All GnRH-FITC containing treating medium should be protected from light and used up within a few h.
13. Preheat the six GnRH-FITC treating medium (prepared in step 1.12) and 4 mL complete medium to 37 °C.
14. Dilute 3 µL of 5 mM fluorescent probe solution (nuclear counterstain mentioned in the Materials List) in 3 mL PBS to 5 µM. This solution should be protected from light, keep it at RT and use it up within a few hours.

2. Confocal Laser Scanning Microscopy (CLSM)

1. GnRH uptake method
   1. Take the first microscopic slide prepared in step 1.6 and pipette out the complete medium from each of the seven well. Add 250 µL of preheated complete medium into the first well. Use this as a negative control. Add 250 µL of the preheated GnRH-FITC treating media (from 1.13) to each of the next six wells (3 wells with 1 µM and 3 with 10 µM). Incubate the slide in a humidified, 5% CO₂ atmosphere incubator at 37 °C for 5 h.
   2. Pipette out the medium from each well and wash the cells with 250 µL PBS. Add 250 µL of fixing solution (10% neutral buffered formalin) into each well and incubate the slide at RT, for 10 min.
   3. Pipette out the fixing solution from each well and wash the cells with 250 µL PBS. Add 250 µL of PBS containing 5 µM fluorescent probe solution prepared in step 1.14 into each well and incubate the slide at RT, for 10 min.
   4. Pipette out the fluorescent probe solution from each well, and wash the cells twice with 250 µL PBS carefully. Add 3-4 drops of mounting medium into each well, finally. Keep the slide in dark at RT, until the imaging.
2. GnRH-I-R expression method
   1. Take the second 8-well microscopic slide prepared in step 1.7 and pipette out the complete medium from each of the five wells.
   2. Add 250 µL of preheated complete medium into the first well, use this as negative control. Add 250 µL of preheated complete medium into the second well too, and use this to examine GnRH-I-R expression before the GnRH treatment.
   3. Add 250 µL each of the three preheated 10 µM GnRH-FITC treating media into the next three wells. Use these wells to pretreat the cells with GnRH-FITC conjugates before examining the GnRH-I-R expression. Incubate the slide in a humidified, 5% CO₂ atmosphere incubator at 37 °C for 1 h.
   4. Pipette out the treating medium from each of the three wells which will be used to examine GnRH-I-R expression after the GnRH treatment and wash these wells with 250 µL of preheated complete medium. Add 250 µL of preheated complete medium into each of the three wells. Incubate the slide in a humidified, 5% CO₂ atmosphere incubator at 37 °C for 1 h.
   5. Pipette out the medium from each of the five wells and wash the cells with 250 µL PBS. Add 250 µL of fixing solution into each well and incubate the slide at RT, for 10 min.
   6. Pipette out the fixing solution from each well and wash the cells with 250 µL of PBS. Add 250 µL of PBS containing 5% bovine serum albumin (BSA) blocking solution into each well and incubate the slide at RT, for 1 h.
   7. Dilute 10 µL of GnRH-I-R primary antibody in 1 mL PBS (1:100 ratio). Keep it at RT and use it up within a few hours.
   8. Pipette out the BSA blocking solution from each well except the negative control (first well). Wash the cells with 250 µL PBS (except the negative control well). Add 250 µL of PBS containing GnRH-I-R primary antibody prepared in step 2.2.7 into each well (except the negative control). Incubate the slide at RT, for 1 h, in a dark place.
   9. Dilute 2.5 µL of Alexa 546 labeled secondary antibody in 1.25 mL PBS (1:500 ratio).
      NOTE: This solution should be protected from light, keep it at RT and use it up within a few hours.
   10. Pipette out solutions from each of the five wells and wash the cells with 250 µL PBS. Add 250 µL of PBS containing AF 546 labeled secondary antibody prepared in step 2.2.9 into each well. Incubate the slide at RT, for 1 h in dark.
   11. Pipette out the solution from each well and wash the cells with 250 µL PBS. Add 250 µL of PBS containing 5 µM fluorescent probe solution prepared in step 1.14 into each well and incubate the slide for 10 min, at RT.
   12. Pipette out the fluorescent probe solution from each well and wash the cells twice with 250 µL PBS carefully. Add 3-4 drops of mounting media into each well, afterwards. Keep the slide in dark at RT until the microscopic imaging.
3. Imaging
   1. Image the cells by inverted confocal laser scanning microscope (using 63X oil immersion objective)
      1. Use the following excitation/emission wavelengths. GnRH conjugates: 488/514 nm, Alexa 546 labeled antibody: 488/546 nm (use only for the GnRH-I-R expression method), Draq5 fluorescent probe: 633/680 nm.
      2. Set up the imaging parameters using the negative control cells. Use these parameters to image treated cells (Figure 3). Re-adjust imaging parameters on each microscopic slide at the beginning of the analysis. Fine-tune and export images with the software provided by the manufacturer if necessary.

3. Fluorescence-activated Cell Sorting (FACS)

1. GnRH quantification method
   1. Take the plate prepared in step 1.8 and pipette out the complete medium from each of the seven wells. Add 1 mL of preheated complete medium into the first well. Use this as a negative control. Add 1 mL each of the six preheated treating media (3 wells with 1 µM and 3 wells with 10 µM) into the next six wells. Incubate the plate in a humidified, 5% CO₂ atmosphere incubator at 37 °C for 5 h.
   2. Pipette out the medium from each well. Wash the cells twice with 2 mL PBS carefully. Add 500 µL of trypsin-EDTA solution into each wells. Incubate the plate at 37 °C until cells detach (approximately 10 min).
   3. Add 1 mL of complete medium into each well to stop trypsin. Shake the plate. Suspend the cells gently with a pipette, and transfer the suspension from each well into FACS tubes. Centrifuge the tubes at 150 x g for 4 min, at 4 °C.
   4. Pour out the supernatants carefully, with one move. Add 500 µL of ice-cold PBS into each FACS tube and gently re-suspend the cells. Keep the tubes on ice until the end of the experiment.
2. Analysis and calculation
   1. Analyze the cells by flow cytometry. For excitation, use 488 nm argon laser wavelength and for detection use 530 nm wavelength. Set up the flow cytometer parameters by running the negative control cells. Use these parameters to analyze treated cells (Figure 4A). Evaluate the data with the manufacturer's software, determine median fluorescent intensity (MFI) values, and calculate the relative MFI values.

Results

Images obtained by confocal laser scanning microscopy (CLSM) offer spectacular information about the uptake of GnRH-FITC conjugates on the given cell culture in a time and concentration dependent manner. In parallel with these GnRH-FITC conjugates, the presence of the GnRH-I-R on the cell surface is also verifiable by the CLSM experiment. Furthermore, by using a far-red DNA staining fluorescent probe, it is possible to counterstain the nuclei of cells besides GnRH and GnRH-I-R. However, w...

Discussion

Experiments described herein use selectively labeled GnRH analogues for screening adherent cell cultures in vitro. Confocal microscopy and flow cytometry methods are suitable for tracking and quantifying the cellular uptake of these GnRH-FITC conjugates in a time and concentration dependent manner. These experiments have the following critical steps: 1) maintain a sterile, healthy cell culture; 2) GnRH peptides must be of high quality; 3) reasonable concentrations and incubation times must be followed; ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EBC-1	JCRB Cell Bank	JCRB0820	Human lung squamous cell carcinoma
BxPC-3	ATCC	CRL-1687	Human pancreatic adenocarcinoma
Detroit-562	ATCC	CCL-138	Human pharyngeal carcinoma
RPMI-1640 Medium with L-glutamine	Lonza	BE12-702F	Culture medium for BxPC-3 cells
Eagle's Minimum Essential Medium	Lonza	BE12-611F	Culture medium for EBC-1 cells, and complemented with 0.1% sodium pyruvate for Detroit-562 cells
Fetal Bovine Serum	Euro Clone	ECS0180L	Complements the culture medium
MycoZap Plus-CL	Lonza	VZA-2012	Complements the culture medium (antibiotics)
Standard Line Cell Culture Flasks	VWR	10062-872	Provide consistent, sterile growth environment for cells
Trypsin-EDTA Mixture	Lonza	BE17-161E	Remove attached cells
Phosphate Buffered Saline (PBS)	Lonza	BE17-516F	Solvent
Dimethyl sulfoxide	Sigma-Aldrich	D8418-100ML	Solvent
Trypan Blue Solution, 0.4%	Thermo Fisher Scientific	15250061	Used to counting cells
[D-Lys6(FITC)]-GnRH-I	made by us	-	Purity ≥98% (HPLC)
[D-Lys6(FITC)]-GnRH-II	made by us	-	Purity ≥98% (HPLC)
[Lys8(FITC)]-GnRH-III	made by us	-	Purity ≥98% (HPLC)
glass bottom 8-well microscopic slide	Ibidi	80826	Use in CLSM experiment
Corning Costar cell culture plate, 12 well	Sigma-Aldrich	CLS3513-50EA	Use in FACS experiment
Fixing solution (10% neutral buffered formalin)	Bio-Optica	01V60P	Fix adherent cells
Bovine Serum Albumin	Sigma-Aldrich	A7906-10G	Prevent the nonspecific binding of the antibodies
GnRHR Antibody	Proteintech	19950-1-AP	Immunocytochemistry reagent, bind to the human type-I GnRH receptors
Secondary Antibody, Alexa Fluor 546 conjugate	Thermo Fisher Scientific	A11035	Immunocytochemistry reagent, bind to the primary antibody
Fluorescent probe solution (Draq5)	Thermo Fisher Scientific	62254	Counterstain nuclei
Fluoromount Aqueous Mounting Medium	Sigma-Aldrich	F4680-25ML	Use in CLSM experiment, preserving fluorescence
Inverted microscope	Elektro-Optika Ltd.	Alpha XDS-2T	Check the phenotype and confluency of cell cultures
Inverted confocal laser scanning microscope	Zeiss	LSM-710	Use in CLSM experiment
Flow cytometer	BD Biosciences	BD FACSCalibur	Use in FACS experiment